Immunostimulating method

ABSTRACT

A method is disclosed for stimulating the immune system of a warm-blooded animal by the production of antibodies by administering an effective amount of Cytophaga allerginae lipopolysaccharide endotoxin.

BACKGROUND OF THE INVENTION

This invention relates to a method of stimulating the immune system of awarm-blooded animal by the production of antibodies.

It is well-known that warm-blooded animals have several types of cellswhich constitute various lines of defense of the body against theinvasion of foreign agents. One line of defense involves the leukocytesand macrophages in the blood stream which are capable of phagocytizingthe foreign agent and often destroying it when such cells come incontact with the foreign agent.

Another line of defense involves an immune mechanism which is broughtinto action by the antigenic constituent of the foreign agent. One typeof immune mechanism involves the B-cells which are precursors of cellsthat secrete immunoglobulins (Igs) or antibodies. The immunoglobulins orantibodies play a significant role in the fight against infection causedby bacteria or other infectious microorganisms.

Consequently, methods of stimulating the immune system to produceantibodies are useful for immune-deficient patients.

It is known that different B-cell mitogens stimulate secretion ofdistinctive patterns of immunoglobulin classes and subclasses in murinesystems. For example, lipopolysaccharides (LPS) stimulate IgM, IgA, IgG3and IgG2b; Corynebacterium parvum stimulates IgM, IgA, IgG1 and IgG3;and pokeweed mitogen stimulates IgM, IgA, IgG1, IgG2a, IgG2b and IgG3.

Bacterial endotoxins are complex molecules that containlipopolysaccharides. Although these endotoxins can stimulate antibodyproduction, they exhibit extreme toxicity which appears to reside intheir lipid fraction. When injected in sufficient amounts, they usuallycause irreversible shock within an hour our two.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a method of stimulating the immunesystem of a warm-blooded animal by the production of antibodies whichcomprises administering to said warm-blooded animal an effective amountof Cytophaga allerginae endotoxin. The use of this method is enhanced bythe relative non-toxicity of the endotoxin in comparison to conventionalendotoxins such as from Salmonella which are known to be highly toxic.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the chromatographic profile of the phenol extract ofCytophaga allerginae from anion exchange chromatography.

DETAILED DESCRIPTION OF THE INVENTION

Cytophaga is a genus of bacteria classified in Bergey's Manual ofDeterminative Bacteria, Buchanan and Gibbons, Eds., 8th ed., 1977,Williams and Wilkins Co., Baltimore, pp. 101-104. Members of the genusare common in soil and in both freshwater and marine environments.Species of the Cytophaga have not generally been shown to initiate humandisease, although the endotoxin of Cytophaga allerginae has beendescribed as a putative agent of occupation related lung disease. SeeFlaherty et al., Infect. Immun. 43(1), 206-216 (1984).

The isolation and characterization of the Cytophaga allerginae speciesare disclosed by Liebert et al., App. Environ. Microbiol. 39, 936-943(1984). An isolate of the organism was obtained from a biomass growingin the demister vanes in a chilled (16° C.) water spray airhumidification system. This chilled-water spray humidification systemwas installed in a textile-producing facility to provide humidity andtemperature control. To humidfy air by this system, ambient outside airwas filtered and drawn through a chilled-water (16° C.) spray. Afterhumidification, large water droplets were removed by passage throughaluminum demister vanes set at angles to the direction of air flow.

An isolate of this species which contains a biologically activeendotoxin, designated WF-164, was made available to the public by beingdeposited without restriction in the permanent collection of theAmerican Type Culture Collection, Rockville, Md., under accession numberATCC 35,408. Samples of this strain can be obtained by the public uponrequest to that depository.

The morphology and culture characteristics of Cytophaga allerginae asdisclosed by Liebert et al., Ibid., are as follows:

The organism was determined to be a gram-negative, elongated, flexiousbacillus. Transmission election microscopy (TEM) negative staining ofwhole mounts with 2.0% ammonium molybdate revealed nonflagellated cellsmeasuring 0.3 by 3.5 to 9.0 μm. After 24 h of growth at room temperature(ca. 23° C.) on nutrient agar (NA), the cells had an average length of3.5 μm; in nutrient broth (NB), the average cell length measured 4.4 μm,although longer cells were observed after being grown in 1% peptonewater. The average cell length was 8.9 μm after 24 h of growth.

The ultrastructure of the organism revealed a bacillus having agram-negative cell envelope with a trilaminar inner membrane(cytoplasmic membrane), an intermediate layer (peptidoglycan), and aconvoluted, trilaminar outer membrane (lipopolysaccharide). There wasfound an increased density of the cell wall in TEM preparations treatedwith ruthenium red. Vesicular tubular structures similar to thoseobserved on the surfaces of Cytophaga johnsonae C4 and Flexibacter sp.strain BH3 were observed on the surfaces of the Cytophaga allerginae.

Gliding and flexing movements were observed by light microscopy on solidsurfaces and in liquid cultures. Typical spreading, fingerlikeprojections of the bacterial colonies were observed on both Cytophagaand vy/2 agars. No fruiting bodies or microcysts were observed. In oldercultures, autolysis and spheroplasts were observed. The organismproduced a bright yellow pigment when grown on all culture media. Theextracted pigment demonstrated a maximum absorption peak at 450 nm.suggesting either a carotenoid or a flexirubin-type pigment. TheKOH-flexirubin test was positive.

The Cytophaga allerginae, originally isolated on plate count (PC) agarat 20° C., also grew at 10 or 30° C. but not at 4 or 37° C. Growth wasobserved within the pH range from 5.5 to 9.5 and in NB with 1.5% NaClbut not with 3.0% NaCl. Growth occurred on eosin methylene blue (EMB)agar but not on MacConkey agar. Anaerobic growth was observed inhalf-strength NB and on PC agar after 48 h. However, noticeably moreluxuriant growth occurred on the same media incubated aerobically.

Of the macromolecules examined, chitin, starch, carboxymethyl cellulose,caesin, and gelatin were hydrolyzed, whereas cellulose, agar, andalginate were not hydrolyzed. Results of the other biochemical tests aregiven in Table 1, below. Of the 11 antibiotics tested, chloramphenicol,carbenicillin and tetracycline were inhibitory to Cytophaga allerginae.

Comparison of the cellular fatty acid content of Cytophaga allerginaewith those of several reference strains revealed a lipid profile similarto those of C. Aquatilis, C. johnsonae, and also Flexibacteraurantiacus. Little difference in the relative concentrations ofstraight- and branch-chain fatty acids of Cytophaga allerginae and thethree reference strains was detected. Methyltetradecenoic acid was themajor lipid (45% of the total fatty acids) detected in all four of thesestrains. However, there were differences in the relative concentrationsof straight- and branched-chain fatty acids of Cytophaga allerginae andthe species of Flavobacterium.

Whole-cell carbohydrate composition demonstrated little similaritybetween Cytophaga allerginae and any of the Flavobacterium referencestrains. Cytophaga allerginae contained a higher percentage ofN-acetyl-D-galactosamine (20.9%) than the reference Cytophaga spp. andFlavobacterium spp. Also, unlike the other reference strains analyzed,the highest percentage of total carbohydrates of Cytophaga allerginaewas composed of two components, heptose and dideoxyhexosamine, whicheluted at the same retention time on packed columns.

Finally, Cytophaga allerginae was determined to have a DNA basecomposition of 34.8 mol% of G+C (mean of five determinations). DNAhomology studies demonstrated a 77.8% similarity of Cytophaga allerginaeto C. aquatilis.

                  TABLE 1                                                         ______________________________________                                        Biochemical Characteristics of Cytophaga allerginae                           Characteristics       Result.sup.a                                            ______________________________________                                        Polysaccharide degradation                                                    Cellulose             -                                                       Carboxymethyl cellulose                                                                             +                                                       Chitin                +                                                       Agar                  -                                                       Alginate              -                                                       Casein                +                                                       Starch                +                                                       Gelatin               +                                                       Carbohydrate utilization.sup.b                                                Glucose oxidation     +                                                       Lactose oxidation     -                                                       Maltose oxidation     +                                                       Cellobiose oxidation  +                                                       Sucrose oxidation     -                                                       Arabinose oxidation   +                                                       Glucose fermentation  -                                                       Other                                                                         Cytochrome oxidase production                                                                       +                                                       Catalase production   +                                                       β-Galactosidase production                                                                     +                                                       Phenylalanine deaminase                                                                             -                                                       Arginine dihydrolase  -                                                       Lysine decarboxylase  -                                                       Ornithine decarboxylase                                                                             -                                                       Urease production     -                                                       DNase production      -                                                       Hydrogen sulfide production                                                                         -                                                       Lecithinase production                                                                              -                                                       Acetylmethycarbinol production                                                                      -                                                       Methyl red test       -                                                       Indole production     -                                                       Nitrate reduction     -                                                       Antibiotic susceptibility (amt).sup.c                                         Ampicillin (10 μg) -                                                       Bacitracin (10 U)     -                                                       Carbenicillin (50 μg)                                                                            +                                                       Cephalothin (30 μg)                                                                              -                                                       Chloramphenicol (30 μg)                                                                          +                                                       Gentamicin (10 μg) -                                                       Neomycin (30 μg)   -                                                       Penicillin (10 U)     -                                                       Polymyxin B (300 U)   -                                                       Streptomycin (10 μg)                                                                             -                                                       Tetracycline (30 μg)                                                                             +                                                       ______________________________________                                         .sup.a +, Positive result: -, negative result                                 .sup.b Production of acid in Board and Holding medium, J. Appl. Bacteriol     23.                                                                           XI-XII(1960).                                                                 .sup.c +, inhibitory: -, not inhibitory to Cytophaga allerginae.         

Isolation and purification of the lipopolysaccharide endotoxin fromCytophaga allerginae can be carried out by methods described by Flahertyet al., Infect. Immun. 43(1), 206-213 (1984), the disclosure of which isincorporated herein by reference. According to these methods, thelipopolysaccharide endotoxin preferably is obtained by a phenol-waterextraction of a culture of the organism followed by purification byanion exchange column chromatography of the extracts.

In a preferred method, the endotoxin was extracted and purified asfollows:

Phenol-Water Extract

Cytophaga allerginae, WF.-164, was grown in nutrient broth (Difco,Detroit, Mich.) at 23° C. for 14 days. The bacteria were recovered fromthe broth by centrifugation and washed twice with 0.85% saline. Thelipopolysaccharide (LPS) fraction was then recovered by using a 5-minphenol-water extraction as described by Westphal and Jann, MethodsCarbohydr. Chem. 5, 83-92 (1965). The water layer was recovered anddialyzed against deionized water for 24 to 48 h at room temperature,using 30 changes of water. After dialysis, the concentration of phenolin the dialysate was determined by UV adsorption at 278 and 283 nm,using a phenol-water calibration curve. The dialysates contained ≦2.0ppm (≦2.0 μg/ml) of phenol. By high-pressure filtration, the LPSfractions were filtered through a 0.45 μm filter (Millipore). The eluatewas then concentrated 10-fold by high-pressure filtration, using Spectrastirred cells with 10,000-molecular weight cutoff filters. The retainedLPS fractions with molecular weights of >10,000 were lyophilized.

Anion Exchange Purification

The LPS recovered by the phenol extraction was fractionated byanion-exchange chromatography using a DEAE-Sepharose CL-6B (PharmaciaFine Chemicals, Inc., Piscataway, N.J.) column (10 by 1.5 cm). Allextracts were equilibrated with the starting buffer, 0.05M potassiumphosphate buffer (pH 7.2) with 0.15M NaCl, and eluted with a stepwisesalt gradient (0.15 to 1.0M NaCl in the phosphate buffer).

The chromatographic profile of the phenol extract of the Cytophagaallerginae while subjected to the aforesaid anion exchange purificationis shown in the accompanying FIG. 1. The solid line denotes absorbance(optical density) at 218 nm; the broken line shows salt gradient.Endotoxin activity resided principally in fraction 1. Conversely,fraction 4 contained one-tenth the endotoxin activity of fraction 1.When compared with an E. coli standard curve, fraction 1 (F-1) had acomputed endotoxin equivalent of 1.21 ng/ml at a dilution of 7.8 ng/mlor 15.5% (wt/wt).

The chemical constituents present in the above column purified fraction(F-1) are shown in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Chemical Constituents of                                                      Cytophaga allerginae endotoxin                                                Constituent        %                                                          ______________________________________                                        Carbohydrate.sup.a                                                            Glyceraldehyde     0.2                                                        Rhamnose           2.8                                                        Fucose             1.1                                                        Ribose             0.1                                                        2-Deoxyribose      0.2                                                        Arabinose          0.1                                                        Mannose            1.4                                                        Galactose          0.6                                                        Xylose             0.1                                                        Glucose            1.9                                                        Heptose            33.9                                                       N--Acetylgalactosamine                                                                           5.1                                                        N--Acetylglucosamine                                                                             18.1                                                       Phosphorus          0.57                                                      RNA                ≦0.15                                               Water              20.0                                                       Lipid.sup.b                                                                   11:0               .sup. --.sup.c                                             12:0               .sup. Tr.sup.d                                             13:0               --                                                         13:1               --                                                         14:0               5.0                                                        20H 14:0           Tr                                                         30H 14:0           Tr                                                         14:1               2.0                                                        15:0               9.0                                                        i15:0              7.0                                                        a15:0              16.0                                                       15:1               --                                                         16:0               23.0                                                       16:1               17.0                                                       17:0               --                                                         i17:0              3.0                                                        a17:0              Tr                                                         17:1               Tr                                                         18:0               3.0                                                        18:1               11.0                                                       19:0               --                                                         19:1               --                                                         20:0               --                                                         ______________________________________                                         .sup.a Results expressed as percentage (weight/weight) of total.              .sup.b Results expressed as percentage of total lipid.                        .sup.c --, Not detected.                                                      .sup.d Tr = ≦2% of total lipid.                                   

The manner of administering the Cytophaga allerginae endotoxin to awarm-blooded mammal can be, for example, by intraperitoneal,subcutaneous, intradermal or intramuscular administration. The endotoxinis thus administered in a pharamceutically acceptable carrier, forexample, sterile water or physiological saline, preferably in amounts offrom about 0.001 to about 1 mg/kg of body weight. Pharmaceuticallyacceptable carriers and their methods of compounding are well-known andcan be selected by reference to a common text such as Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 11th edition,1980.

LD₅₀ Determinations

The LD₅₀ of Cytophaga allerginae lipopolysaccharide endotoxin isolatedby the above phenol extraction method and purified by the foregoinganion-exchange chromatography method was determined as follows:

The animals used in this determination were male Cr1: CD-1 (ICR) BRalbino mice obtained at 28 days of age from Charles River BreedingLaboratory, Portage, Mich. The mice were subjected to routine quarantineprocedures for 10 days and were released in good condition. The micewere double-housed until put on test at which time they weresingle-housed in stainless steel, suspended cages. They were providedfood (Ralston Purina Certified Rodent Chow #5002) and water ad libitum.

The two test materials were identified as Salmonella typhosa 0901,Control #688706, lot #3124-25 and Cytophaga allerginae, B.I. 164 (no lot#) endotoxins. The various dosages were prepared by dilutingconcentrated solutions with physiological saline (0.9% Sodium ChlorideInjection USP, American Hospital Supply Corp., Irvine, CA 97714, lot#B4L874C). Injections were done intraperitoneally using a syringe with a23 ga×1" needle. Each mouse was given a single dose injection withvolumes ranging from 0.2 to 0.6 ml, and was observed at least twicedaily for two weeks. Dead mice were removed and discarded withoutnecropsy.

Initial dosages were based on previous experience (Cytophaga--100μg/mouse killed 2 of 4) or literature (Salmonella typhosa--range of 10³to 10⁶ μg/kg was an approximate LD₅₀). At each of the following dosages(mg/kg) five mice were innoculated:

    ______________________________________                                        Cytophaga     Salmonella                                                      ______________________________________                                        40            10.sup.3                                                        20            10.sup.6                                                        10            6.7 × 10.sup.7                                            5             3.3 × 10.sup.7                                            2.5           10.sup.9                                                        1.25                                                                          ______________________________________                                    

Early indications were that the mice injected with Salmonella endotoxinwere not ill, so another set of five mice per level were injected withfresh solutions. This time, however, 50 mgs of lypholized material wasweighed out rather than 1 mg. This necessitated two additional dilutionsand one mouse was injected with a quantity of each of these "stock"solutions (1 at 50 mg/kg and 1 at 1 mg/kg). The former mice died whilenone of the mice at 1 mg/kg or lower died. Also, an insufficient numberof the mice injected with the Cytophaga endotoxin died to permit asatisfactory estimate of the dose-response. Therefore, the final seriesof dose levels (mg/kg) were escalationed to ascertain the dose-responsecurve:

    ______________________________________                                        Cytophaga     Salmonella                                                      ______________________________________                                        240           100                                                             160           50                                                              80            20                                                                            5                                                               ______________________________________                                    

The results were as follows:

    ______________________________________                                        Cytophaga allerginae                                                                           Salmonella typhosa                                           Dose   # dead/# injected                                                                           Dose      # dead/# injected                              ______________________________________                                        240    5/5           100       5/5                                            160    5/5            50       5/5                                            80     4/5            20       4/5                                            40     2/5            5        5/5                                            20     0/5            1        0/1                                            10     1/5           10.sup.3  0/10                                            5     0/5           10.sup.6  0/10                                           2.5    0/5           6.7 × 10.sup.7                                                                    0/10                                           1.25   0/5           3.3 × 10.sup.7                                                                    0/10                                                                10.sup.9  0/10                                           ______________________________________                                    

Computer calculated probit responses based on the above results(superfluous dose levels with zero responses were not entered) were asfollows:

    ______________________________________                                                      Salmonella typhosa*                                             Cytophaga allerginae        Results                                           LD Value                                                                              Results (mg/kg)                                                                           95% C.I.    (mg/kg) 95% C.I.                              ______________________________________                                        90      116.20      67.188-462.14                                                                             1.921                                         50      40.735      22.535-71.638                                             10      14.280       3.352-25.039                                             ______________________________________                                         *The only obtainable estimate based on the responses (Salmonella) was fro     nonlinear interpolation using the binominal method.                      

The foregoing LD₅₀ determinations show that the Cytophaga allerginaelipopolysaccharide endotoxin has an LD₅₀ of about 40 compared to theLD₅₀ of about 2 for the Salmonella typhosa endotoxin.

Immunostimulant Activity

The induction of de novo human immunoglobulin synthesis--IgG, IgA andIgE--by Cytophaga allerginae lipopolysaccharide endotoxin (CAE) isolatedand purified by the foregoing method was determined by the standardpokeweed mitogen stimulated immunoglobulin synthesis method. Thisstandard method is described, for example, by Saxon, Manual of ClinicalImmunology, Eds. Rose and Friedman, ASM, Washington, D.C., SecondEdition, 1980, pp. 151-156; and Sampson and Buckley, J. Immunol. 127,829-834 (1981). According to this method, the effect of pokeweed mitogenon human peripheral blood lymphocytes cultured in complete media isdetermined. The stimulating effect of the CAE is then assessed by addingvarying concentrations of the CAE to the media instead of the pokeweedmitogen and comparing the respective results.

In order to determine whether CAE can induce immunoglobulin synthesis byhuman peripheral blood lymphocytes (PBL) in vitro, human PBL werepurified and cultured with either pokeweed mitogen (PWM) at 2.5 and 5μg/ml, or CAE (1.25-20 μg/ml) for six to ten days. Following thatincubation, the supernatants were harvested and the levels of thedifferent immunoglobulins (IgG, IgA and IgE) were determined by eitherthe alkaline phosphatase ELISA assay (IgG and IgA) or the Avidin-BiotinELISA assay (IgE).

ELISA (enzyme linked immunosorbent assay) is a conventional serologyassay technique originally introduced by Engvall and Perlman,Immunochem. 8, 871-874 (1971) and J. Immunol.. 109, 129-135 (1972). Ituses enzyme markers conjugated to antigen or antibody instead ofradioactive isotopes or fluorescent dyes and is well adapted to thequantitative assay of immunoglobulins, e.g. IgG. The enzymatic activityis detectable with conventional photometric instrumentation. That is,the antigen or antibody complex can be detected by the enzyme labelchanging the color of an added substrate. The optical density of thefinal color is directly proportional to the amount of the unknownantibody or antigen in the original test solution. In the alkalinephosphatase method, that enzyme is comjugated to the antigen (e.g., IgG)such as by the use of glutaraldehyde, and the added substrate isp-nitrophenyl phosphate which is hydrolyzed to p-nitrophenol having ayellow color that can be measured at 405 nm. Further backgroundinformation on this assay can be had by reference to Voller, Bidwell andBartlett, "The Enzyme Linked Immunosorbent Assay (ELISA)," DynatechLaboratories, Inc., Alexandria, Va.

The ELISA determinations were carried out as follows:

Immulon II plates were coated with optimal concentrations of affinitypurified antisera to human immunoglobulins (IgG, IgA and IgE). Afterincubation overnight at 4° C., the plates were washed with phosphatebuffered saline (PBS). Unbound reactive sites were blocked with 1%bovine serum albumin in PBS. Following incubation at room temperaturefor one hour, plates were washed with PBS. Standard concentrations ofimmunoglobulins, controls and test samples were added to the plates,incubated for two hours at 37° C. and washed with PBS. Two secondaryamplification methods were used to detect and quantitate immunoglobulinclasses or isotypes.

(1) Alkaline Phosphatase ELISA: Optimal concentrations of anti-humanimmunoglobulin isotype linked to alkaline phosphatase was added to theplates. The plates were incubated at 37° C. for two hours and washedwith PBS. Dinitrophenyl phosphate substrate was added and the platesincubated in the dark for 30 minutes at room temperature. The reactionwas terminated with NaOH. The absorbance at 405 nm was determined foreach well.

(2) Biotin-Avidin ELISA: Optimal concentrations of biotin conjugatedanti-human immunoglobulin isotype were placed in the plate. Followingincubation for two hours at 37° C., the plates were washed with PBS.Optimal concentrations of Avidin D horse radish peroxidase (VectorLaboratories, San Francisco, Calif.) were added to the plates, incubatedfor 30 minutes at 37° C. and washed with PBS. O-phenylene diaminesubstrate (Zymed Labs, San Francisco, Calif.) was added to the plates.The plates were incubated for 30 minutes at room temperature in thedark. The absorbance was determined at 492 nm.

The assay results set forth in Tables 3 to 5, below, indicate that CAEadded to cultures in vitro induces IgG, IgA and IgE synthesis by humanPBL. In these tables, the IgG and IgA is reported as nanograms/ml ofantibody produced while IgE is reported as picograms/ml of antibodyproduced. It is seen that CAE induces IgG and IgA synthesis comparableto or greater than the synthesis induced by the commonly used PWMmitogen. CAE also induces de novo IgE synthesis in seven out of tendonors while PWM induces de novo IgE synthesis in four out of tendonors. In summary, CAE is a useful immunostimulant which can inducehuman IgG, IgA and IgE synthesis in vitro (see Tables 3, 4 and 5). Inaddition, CAE cultured with human PBL for 6 days induces fibroblast likecells which differ from the morphology of PBL incubated with PWM for sixdays.

                                      TABLE 3                                     __________________________________________________________________________    De novo IgG synthesis                                                         ng/ml                                                                         μg/ml                                                                             Donor #                                                                Mitogen                                                                              1    2  3  4  5  6    7  8    9    10                                  __________________________________________________________________________    PWM 5  1,940                                                                              380                                                                              420                                                                              960                                                                              140                                                                              >20,000                                                                            680                                                                              >20,000                                                                            >20,000                                                                            >20,000                                 2.5                                                                              2,540                                                                              340                                                                              640                                                                              280                                                                              200                                                                              >20,000                                                                            1160                                                                             >20,000                                                                            >20,000                                                                            >20,000                             CAE 20 280  240                                                                              380                                                                              180                                                                              3060                                                                             5,400                                                                              2500                                                                             16,760                                                                             12,880                                                                             >20,000                                 10 0    60 640                                                                              340                                                                              1860                                                                             2,000                                                                              840                                                                              16,160                                                                             3,680                                                                              >20,000                                 5  0    80 620                                                                              240                                                                              3460                                                                             720  340                                                                              >20,000                                                                            3,080                                                                              >20,000                                 2.5                                                                              0    60 140                                                                              120                                                                              2160                                                                             700  1440                                                                             15,200                                                                             1,560                                                                              >20,000                                 0.25                                                                             0    100                                                                              420                                                                              0  1660                                                                             600  680                                                                              18,160                                                                             1,040                                                                              15,000                              None   20   134                                                                              200                                                                              140                                                                              2060                                                                             0    350                                                                              4,960                                                                              150  5,900                               Freeze 260  160                                                                              520                                                                              400                                                                              540                                                                              0    400                                                                              240  320  200                                 Thaw                                                                          5 ×                                                                     __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    IgA Synthesis                                                                 ng/ml                                                                         ug/ml   Donor #                                                               Mitogen 1    2 3  4  5  6   7  8  9  10                                       __________________________________________________________________________    PWM  5  39.5 32                                                                              40 23 42.2                                                                             >1000                                                                             48.6                                                                             660                                                                              935                                                                              482                                           2.5                                                                              23.5 34                                                                              44 19 35.6                                                                             640 90.6                                                                             260                                                                              775                                                                              242                                      CAE  20 1.5  17                                                                              56 13 66.4                                                                             93  74.6                                                                             180                                                                              145                                                                              272                                           10 0    4 40 17 62.6                                                                             76  44.6                                                                             240                                                                              205                                                                              272                                           5  2.5  8 60 21 67.6                                                                             0   40.6                                                                             380                                                                              185                                                                              26                                            2.5                                                                              4.5  6 40 15 105.6                                                                            0   70.6                                                                             270                                                                              125                                                                              10                                            1.25                                                                             0    5 ND 27 55.6                                                                             0   33.6                                                                             240                                                                              38 47                                       None    2.5  3 ND ND 66.0                                                                             39  40.6                                                                             460                                                                              80 135                                      Freeze &                                                                              2.5  0 8  27 4.4                                                                              0   3.4                                                                              0  25 18                                       Thaw                                                                          5 ×                                                                     __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    De Novo IgE Synthesis (picograms/ml)*                                         Mitogen                                                                       (μg/ml)                                                                           1.sup.a                                                                           2.sup.a,b                                                                          3.sup.b                                                                           4.sup.b                                                                           5.sup.b                                                                           6.sup.b                                                                            7.sup.a,b                                                                         8.sup.a                                                                           9   10.sup.b                         __________________________________________________________________________    PWM 5  1,750                                                                             2,500                                                                              3,750                                                                             250 2,750                                                                             2,500                                                                              1,600                                                                             2,000                                                                             <100                                                                              50                                   2.5                                                                              1,750                                                                             2,500                                                                              2,750                                                                             250 2,500                                                                             3,500                                                                              2,500                                                                             1,950                                                                             <100                                                                              ND                               CAE 20 <100                                                                              12,250                                                                             8,250                                                                             9000                                                                              4,000                                                                             20,500                                                                             1,300                                                                             1,700                                                                             200 300                                  10 <100                                                                              10,000                                                                             7,500                                                                             5000                                                                              2,250                                                                             13,000                                                                             450 1,100                                                                             <100                                                                              100                                  5  <100                                                                              3,750                                                                              2,250                                                                             7500                                                                              2,250                                                                             65,000                                                                             300 900 200 <50                                  2.5                                                                              <100                                                                              3,250                                                                              2,500                                                                             8000                                                                              2,250                                                                             3,000                                                                              50  900 100 <50                                  1.25                                                                             <100                                                                              6,750                                                                              3,500                                                                             2000                                                                              3,750                                                                             4,250                                                                              <50 700 <100                                                                              <50                              None   <100                                                                              750  4,000                                                                             250 2,500                                                                             11,500                                                                             100 1,100                                                                             200 <50                              5 ×                                                                            100 <100 2,250                                                                             750 150 100  1,200                                                                             100 650 300                              Freeze                                                                        and                                                                           thaw.sup.c                                                                    __________________________________________________________________________     .sup.a PWM stimulates de novo IgE synthesis from PBL of 4 donors.             .sup.b CAE stimulates de novo IgE synthesis from PBL of 7 donors.             .sup.c Values of 5 × freeze and thaw cells cultured for 10 days.        *Background IgE release were subtracted from each value.                      ND Not determined                                                        

The induction of de novo human IgG subclass synthesis--IgG₁ and IgG₂--by Cytophaga allerginae lipopolysaccharide endotoxin in human PBL wasdetermined by the method of Scott and Nahm, J. of Immunol 135:2454-2460(1984). The data in Table 6, below, show that the CAE is betterat stimulating IgG production when compared to an E. coli endotoxin(Gibco). Moreover, the CAE was twice as active at high concentrations.

                                      TABLE 6                                     __________________________________________________________________________    Comparison of E. coli LPS with CAE                                                      IgM        IgG        IgG1       IgG2                               LPS   None                                                                              E. coli                                                                           CAE                                                                              None                                                                              E. coli                                                                           CAE                                                                              None                                                                              E. coli                                                                           Cae                                                                              None                                                                              E. coli                                                                            CAE                           __________________________________________________________________________    Donor                                                                             A <1.5*                                                                             1.1 2  2   1.1 2.4                                                                              --  0.42                                                                              0.69                                                                             --  0.23 0.42                              B 0.7 2.2 4.6                                                                              2   2.8 4  --  1.4 2  --  1.1  1.9                               C 0.2 <1  1.1                                                                              0.25                                                                              0.8 1.5                                                                              --  0.9 1.35                                                                             --  <0.28                                                                              <0.28                         __________________________________________________________________________     *Units are μg/ml                                                           **Gibco E. coli LPS was used at 1/100 dilution and CAE was used at 50         μg/ml.                                                                

Various other examples will be apparent to the person skilled in the artafter reading the disclosure hereof without departing from the spiritand scope of the invention, and it is intended that all such examples beincluded in the scope of the appended claims. Thus, it will beappreciated, for example, that mutants and variants of the Cytophagaallerginae, ATCC 35,408, can be used as sources of the endotoxin toprovide similar immunostimulation results.

What is claimed is:
 1. The method of stimulating the immune system of awarm-blooded animal with a bacterial endotoxin of reduced toxicity toproduce an increased level of antibodies selected from the groupconsisting of IgG, IgA and IgE which comprises administering to saidwarm-blooded animal an effective amount of Cytophaga allerginaelipopolysaccharide endotoxin with a pharmaceutically acceptable carrier,said reduced toxicity being determined by comparison with the toxicityproduced by a Salmonella typhosa endotoxin control and said increasedlevel of antibodies being determined by comparison with the antibodylevel produced by the stimulating effect of an E. coli endotoxin orpokeweed mitogen control on peripheral blood lymphocytes.
 2. The methodof claim 1 in which the IgG antibodies are IgG₁ and IgG₂.